US7842500B2 - Anti-heparan sulfate antibody, method for detection of heparan sulfate, and kit for detection of heparan sulfate - Google Patents

Anti-heparan sulfate antibody, method for detection of heparan sulfate, and kit for detection of heparan sulfate Download PDF

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US7842500B2
US7842500B2 US11/909,956 US90995606A US7842500B2 US 7842500 B2 US7842500 B2 US 7842500B2 US 90995606 A US90995606 A US 90995606A US 7842500 B2 US7842500 B2 US 7842500B2
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antibody
nah
reactivity
solution
reaction
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US20090136964A1 (en
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Kiyoshi Suzuki
Takeshi Ishimaru
Koji Yamamoto
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Seikagaku Corp
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G01N2400/38—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence, e.g. gluco- or galactomannans, Konjac gum, Locust bean gum or Guar gum
    • G01N2400/40—Glycosaminoglycans, i.e. GAG or mucopolysaccharides, e.g. chondroitin sulfate, dermatan sulfate, hyaluronic acid, heparin, heparan sulfate, and related sulfated polysaccharides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/975—Kit

Definitions

  • the present invention relates to a novel anti-heparan sulfate antibody which reacts with N-acetylheparosan and heparan sulfate derived from bovine kidney but does not substantially react with heparan sulfate derived from a mouse Engelbreth-Holm-Swarm tumor tissue, a method of detecting heparan sulfate using the same, and a kit for detecting heparan sulfate.
  • CDSH Completely desulfated-N-acetylated HEP
  • CS-A(S) Chondroitin sulfate A derived from shark
  • CS-A(W) Chondroitin sulfate A derived from whale
  • EHS-HS Heparan sulfate derived from Engelbreth-Holm-Swarm tumor tissue
  • GAG Glycosaminoglycan
  • HSPG Proteoglycan-heparan sulfate
  • NDST Glucosaminyl N-deacetylase/N-sulfotransferase
  • D-glucosamine residue is represented as GlcNH 2 ; hexuronic acid residue is represented as HexA; N-sulfated-D-glucosamine residue is represented as GlcNS; D-glucuronic acid residue is represented as GlcA; N-acetyl-D-glucosamine residue is represented as GlcNAc; and partially de-acetylated NAH is represented as PDNAc-NAH.
  • HS and HEP which are kinds of GAGs, are acidic polysaccharides having a disaccharide-repeated structure containing a D-glucosamine residue and a hexuronic acid (GlcA or IdoA) residue as a basic sugar chain structure. In addition, they are modified by O-sulfation, N-sulfation, or N-acetylation to various degrees.
  • HS is present as HSPG in cell surfaces of almost all animals (Non-patent Document 1), and is a major component of an extracellular matrix.
  • HEP is localized in a mucosal and a granule in a mast cell.
  • HEP-binding proteins bioactive proteins
  • HBPs bioactive proteins
  • HEP is considered to stabilize HBPs such as fibroblast growth factors, hepatocyte growth factor, etc., and to act as a cofactor or a co-receptor, thereby controlling cell proliferation.
  • HEP is considered to stabilize HBPs such as fibroblast growth factors, hepatocyte growth factor, etc., and to act as a cofactor or a co-receptor, thereby controlling cell proliferation.
  • HEP is considered to stabilize HBPs such as fibroblast growth factors, hepatocyte growth factor, etc., and to act as a cofactor or a co-receptor, thereby controlling cell proliferation.
  • a major ligand of HBPs in vivo is considered to be not HEP but HS. That is, HS is considered to regulate the above-mentioned various biological reactions. Therefore, HS that is widely present in animal bodies and has various functions is important component for animals and human. From biological and medical points of view, it is very important to acquire qualitative and quantitative method for precisely detecting HS. Accordingly, a monoclonal antibody against HS may be a unique detection reagent.
  • mAb HepSS-1 and mAb F58-10E4 which are monoclonal antibodies that recognize an N-sulfated glucosamine unit of HS (-[HexA-GlcNS]-), are commercially available and widely used.
  • HS structure of HS is analyzed in accordance with the method of “2.8 Structural analysis using degrading enzyme of glycosaminoglycan and HPLC in combination” described in Non-patent Document 2, it is easily understood that a major component disaccharide of HS is a nonsulfated disaccharide, GlcA-GlcNAc.
  • the ratio reaches 50 to 60% although it depends on an organ from which HS is isolated. That is, the major structure of HS is an N-acetylglucosamine unit (-[GlcA-GlcNAc]-).
  • Non-patent Document 3 NDST activity that catalyzes a first step of the sulfation reaction of HS and HEP is decreased, resulting in increase in the ratio of an N-acetylglucosamine unit.
  • HS derived from kidney is known to contain a glucosamine unit (-[GlcA-GlcNH 2 ]-) (Non-patent Document 4). Therefore, in order to distinguish HS derived from kidney from HSs derived from other organs, it is desirable to use an antibody that specifically recognizes a structure present in HS derived from kidney, that is, a glucosamine unit. Accordingly, detection/quantification of the N-acetylglucosamine unit or glucosamine unit, which forms a nonsulfated domain of HS, is as important as detection/quantification of a sulfated domain.
  • mAb JM-403 which recognizes a glucosamine unit of HS, has been prepared from a mouse that was immunized with HSPG purified from rat glomerulus by van den Born, J. et al. van den Born, J. et al. have reported that HS contains 2 to 3 glucosamine units in average, and that the reactivity of the antibody disappears by acetylation of HS. They have also reported that the antibody does not react with NAH but react with de-N-acetylated NAH more strongly as compared to HS.
  • Non-patent Document 8 discloses that an N-sulfated glucosamine unit present in HS does not affect the reactivity of the antibody, but an O-sulfated structure or an IdoA structure inhibits the reactivity of the antibody.
  • mAb 865 which recognizes an N-acetylglucosamine unit of HS, was originally obtained as an antibody against enterobacterial common antigen (anti-ECA) by Peters, H. et al., and is cross-reactive to a capsular polysaccharide of Escherichia coli K5 (hereinafter, referred to as “NAH”).
  • NAH enterobacterial common antigen
  • van den Born, J. et al. has reported that the antibody does not react with sulfated NAH or N-deacetylated NAH and its epitope is an N-acetylglucosamine unit of 18 sugars or more.
  • Non-patent Document 7 Non-patent Document 7
  • mouse EHS-HS which has high sulfation degree as compared to normal HS.
  • Mouse EHS-HS is HS with high sulfation degree. Therefore, an antibody capable of reacting with mouse EHS-HS reacts with HS of high sulfation degree, so the antibody is considered to have relatively low specificity to the antigen. As described above, an antibody that recognizes nonsulfated region containing N-acetylglucosamine units, glucosamine units, and the like of HS but does not react with mouse EHS-HS was not known, and therefore such an antibody has been desired.
  • Non-patent Document 1 Biochemistry, 10, 20 1445 (1971)
  • Non-patent Document 2 Shin Seikagaku Jikken Koza 3, Carbohydrates II (published by Tokyo Kagaku Dojin Co., Ltd., 1991) p. 49-62
  • Non-patent Document 3 Diabetes, 40, 1449 (1991)
  • Non-patent Document 4 Kidney Int., 41, 115 (1992)
  • Non-patent Document 5 J. Biol. Chem., 270, 31303 (1995)
  • Non-patent Document 6 Infect. Immun., 50, 459 (1985)
  • Non-patent Document 7 J. Biol. Chem., 271, 22802 (1996)
  • Non-patent Document 8 J. Biol. Chem., 270, 31303 (1995)
  • An object of the present invention is to provide an antibody that can be suitably used in detection of HS or NAH in a sample.
  • an object of the present invention is to provide an antibody that specifically recognizes nonsulfated region consisting of N-acetylglucosamine units or glucosamine units.
  • the inventors of the present invention attempted to produce a novel anti-GAG antibody using a substance prepared by binding a protein to NAH as an antigen.
  • NAH which has been used by the inventors of the present invention to produce the antigen is an acidic polysaccharide mainly composed of an N-acetylglucosamine unit (Eur. J. Biochem., 116, 359-364 (1981)).
  • the structure of NAH prepared in accordance with “N-acetylheparosan fraction and method for producing the same” described in JP 2004-018840 A was analyzed by the method “Structural analysis using degrading enzyme of glycosaminoglycan and HPLC in combination” described above.
  • the inventors of the present invention considered that the prepared NAH has excellent properties as an antigenic substance for producing an antibody against NAH and an antibody against N-acetylglucosamine units or glucosamine units of HS.
  • the inventors of the present invention have made extensive studies and have succeeded in producing an antibody that reacts with NAH and an antibody that recognizes N-acetylglucosamine units or glucosamine units of HS. Further, they have discovered that the antibody does not substantially react with mouse EHS-HS, thereby completed the present invention.
  • the present invention provides the followings:
  • the antibody according to any one of (1) to (7) which is a monoclonal antibody produced by a hybridoma formed by cell fusion of: a lymphocyte that is derived from a mammal immunized with a substance obtained by binding a protein to N-acetylheparosan as an antigen; and a myeloma cell derived from a mammal.
  • a monoclonal antibody which is produced by the hybridoma having Deposition number of FERM BP-10534, FERM BP-10535, or FERM BP-10536.
  • a substance which is obtained by chemically binding a protein to N-acetylheparosan, and has antigenicity that enables production of an antibody which reacts with N-acetylheparosan.
  • a hybridoma which is formed by cell fusion of: a lymphocyte that is derived from a mammal immunized with a substance obtained by chemically binding a protein to N-acetylheparosan as an antigen; and a myeloma cell derived from a mammal.
  • a hybridoma which has the Deposition number of FERM BP-10534, FERM BP-10535, or FERM BP-10536.
  • a kit for detecting heparan sulfate or N-acetylheparosan in a sample comprising at least one antibody according to any one of (1) to (11).
  • FIG. 1 A diagram showing a method of producing the modified HEPs.
  • FIG. 2 A first diagram showing the reactivity of NAH33 antibody, NAH43 antibody, and NAH46 antibody to GAGs.
  • FIG. 3 A second diagram showing the reactivity of NAH33 antibody, NAH43 antibody, and NAH46 antibody to GAGs.
  • FIG. 4 A third diagram showing the reactivity of NAH33 antibody, NAH43 antibody, and NAH46 antibody to GAGs.
  • FIG. 5 Diagrams showing specific the reactivity of NAH33 antibody (A), NAH43 antibody (B), and NAH46 antibody (C).
  • FIG. 6 First diagrams showing the reactivity of NAH33 antibody, NAH43 antibody, and NAH46 antibody against different concentrations of Bi-GAGs.
  • the graphs (a) and (b) represent the reactivity of NAH33 antibody
  • the graphs (c) and (d) represent the reactivity of NAH43 antibody
  • the graphs (e) and (f) represent the reactivity of NAH46 antibody.
  • FIG. 7 Second diagrams showing the reactivity of NAH33 antibody, NAH43 antibody, and NAH46 antibody against different concentrations of Bi-GAGs.
  • the graph (a) represents the reactivity of NAH33 antibody
  • the graph (b) represents the reactivity of NAH43 antibody
  • the graph (c) represents the reactivity of NAH46 antibody.
  • FIG. 8 Diagrams (photographs) showing the results of immunohistochemical staining of rat kidney frozen sections using NAH33 antibody (A), NAH43 antibody (B), and NAH46 antibody (C). The arrows show positive sites.
  • FIG. 9 Diagrams (photographs) showing the results of immunohistochemical staining in the case of heparitinase digestion treatment (B, D, and F) and in the case of no heparitinase digestion treatment (A, C, and E).
  • the photographs A and B show the results of staining using NAH33 antibody
  • the photographs C and D show the results of staining using NAH43 antibody
  • the photographs E and F show the results of staining using NAH46 antibody.
  • the solid line arrows represent positive sites.
  • the dashed line arrows represent sites where positive reactions disappeared.
  • FIG. 10 A first diagram showing the detection of bovine kidney HS using antibody-immobilized plates ((i), (ii), and (iii) represent the combinations of the immobilized antibodies and HRP-labeled antibodies shown in Table 4.)
  • FIG. 11 A second diagram showing the detection of bovine kidney HS using antibody-immobilized plates ((i), (iv), and (v) represent the combinations of immobilized antibodies and HRP-labeled antibodies shown in Table 4.)
  • FIG. 12 Diagrams showing the results of detection of HS on cell surfaces by the flow cytometry method.
  • the symbol (+) represents the use of HS-negative cells (heparitinase I digestion), while the symbol ( ⁇ ) represents the use of HS-positive cells (no heparitinase I digestion).
  • the graph (A) shows the reactivity of the negative control antibody to HS-positive cells
  • the graph (B) shows the reactivity of NAH46 antibody to HS-positive cells
  • the graph (C) shows the reactivity of NAH46 antibody to HS-negative cells.
  • the present invention provides antibodies that react with NAH and bovine kidney HS but does not substantially react with EHS-HS (hereinafter, referred to as “antibody of the present invention”).
  • NAH is a capsular polysaccharide isolated from Escherichia coli K5 and an acidic polysaccharide having an N-acetylglucosamine unit as a basic sugar chain structure (Eur. J. Biochem., 116, 359-364 (1981)). NAH can be prepared by, for example, the method described in Reference Example 1 below.
  • the bovine kidney HS is HS in which about 35% of N-positions of D-glucosamine residues are sulfated.
  • Such bovine kidney HS can be purified by treating acetone-defatted bovine kidney with alkaline extraction, protease digestion, removal of nucleic acids, alcohol fractionation, and chondroitinase ABC treatment, followed by chromatography in accordance with the method by Schiller, S. et al. (Lanber, A. et al., Biochim. Biophys. Acta., 29, 443 (1958), Schiller, S. et al., J. Biol.
  • bovine kidney HS includes the HS of research reagent, code number 400700, manufactured by Seikagaku Corporation.
  • Mouse EHS-HS (heparan sulfate derived from an Engelbreth-Holm-Swarm tumor tissue) is HS isolated from mouse EHS (Engelbreth-Holm-Swarm) tumor tissue. Preparation of HS from mouse EHS tumor tissue can be performed in accordance with the method described in JP 07-53756 B, for example. As shown in the results of the analysis of disaccharide compositions described in Reference Example 3 below, such EHS-HS is HS in which about 65% of N-positions of D-glucosamine residues are sulfated.
  • reaction means an immunological reaction or an antigen-antibody reaction
  • the reactivity of an antibody to an antigen can be determined by, for example, the ELISA method, RIA method, plaque method, agglutination method, flow cytometry method, histological staining method, Western blotting method, etc.
  • a reaction signal of the ELISA method using an antibody is enhanced in proportion to an increase in the concentration of an antigen, the antibody is considered to react with the antigen.
  • the phrase “does not substantially react” means that the reactivity of an antibody to an antigen provides no reaction signal or provides only an extremely slight reaction signal by the aforementioned method of determining the reactivity.
  • the reactivity of the antibody of the present invention to EHS-HS when the reactivity to EHS-HS is examined by setting conditions including reaction time, detection method, and concentration of an antigen in an antigen solution and concentration of an antibody in an antibody solution to be used for determination to the same conditions for sufficiently detecting the reactivity of the antibody of the present invention to NAH so as to achieve an absorbance difference of about 2.0 (the absorbance difference can be obtained by subtracting an absorbance obtained in a case of using a blank solution not containing the antibody of the present invention as a test solution from an absorbance in a case of determining the reactivity of the antibody of the present invention to the antigen using a solution containing the antibody of the present invention as a test solution.
  • the reactivity of the antibody of the present invention to EHS-HS is 10% or less, more preferably 5% or less compared to that to NAH, the antibody is considered not to substantially react with EHS-HS.
  • Specific examples of the conditions for detection may be any of the conditions for determination of reactivity of NAH33 antibody, NAH43 antibody, and NAH46 antibody in Example 4 shown below, which can be referred.
  • the ratio of the reactivity of the antibody of the present invention against NAH to the reactivity against other GAGs or the like such as a ratio of the reactivity against NAH to the reactivity against bovine kidney HS and a ratio of the reactivity against NAH to the reactivity against about 40% de-N-acetylated NAH
  • Specific examples of the conditions in the detection may be any of the conditions in determination of reactivity of NAH33 antibody, NAH43 antibody, and NAH46 antibody in Example 4 shown below, which can be referred.
  • the antibody of the present invention is NAH33 antibody, NAH43 antibody, or NAH46 antibody
  • methods of examining the reactivity of the antibody of the present invention to EHS-HS and the ratios of the reactivity of the antibody of the present invention against NAH to the reactivity against other GAGs or the like may be the method described in Example 4 shown below, which can be referred.
  • the antibodies of the present invention are not particularly limited as long as they react with NAH and bovine kidney HS and do not substantially react with EHS-HS. Therefore, the antibodies of the present invention may react with both NAH and HS derived from other than bovine kidney.
  • HS that reacts with the antibody of the present invention contains a nonsulfated disaccharide obtained by the methods described in Examples shown below at a rate of preferably not less than about 40%, more preferably not less than about 50%.
  • the ratio of the reactivity of the antibody of the present invention against NAH to the reactivity of the antibody of the present invention against bovine kidney HS is preferably 10:2 to 10:20, more preferably 100:20 to 100:90.
  • antibody examples include the following antibodies (i) and (ii).
  • an antibody that achieves the ratio of 100:35 to 100:45 include the monoclonal antibodies produced by the hybridoma having Deposition Nos. FERM BP-10534 and FERM BP-10536 (also referred to as “NAH33 antibody” and “NAH46 antibody”, respectively).
  • Specific examples of such an antibody include the monoclonal antibody produced by the hybridoma having Deposition No. FERM BP-10535 (also referred to as “NAH43 antibody”).
  • the antibodies of the present invention are preferably antibodies that do not substantially react with HA.
  • the HA examples include the HA described in Examples shown below.
  • the antibodies of the present invention are preferably antibodies that do not substantially react with HEP or desulfated HEPs, more preferably antibodies that do not substantially react with HEP and desulfated HEPs.
  • the HEPs include HEPs described in Examples shown below.
  • the desulfated HEPs are described in Examples shown below and selected from the group consisting of 6DSH, NSH, NH 2 —HEP, NH 2 -2SH, NH 2 -6SH, NH 2 —CDSH, NAc-HEP, 2SH, 6SH, CDSH, Ac-6DSH, and Ac-NSH. Further, the antibodies are preferably ones that do not substantially react with 2DSH and/or Ac-2DSH.
  • the antibody of the present invention is preferably one that does not substantially react with GAG selected from the group consisting of Ch, CS-A, CS-B, CS-C, CS-D, CS-E, and KS, more preferably one that does not substantially react with Ch, CS-A, CS-B, CS-C, CS-D, CS-E, and KS.
  • GAGs include GAGs described in Examples shown below.
  • the antibody of the preset invention preferably has specific reactivity to de-N-acetylated NAH.
  • Examples of the antibody include the following antibodies (iii) to (v).
  • an antibody that does not substantially react with about 40% de-N-acetylated NAH is preferably one that does not substantially react with about 20% de-N-acetylated NAH, more preferably one that does not substantially react with about 10% de-N-acetylated NAH.
  • Specific examples of such an antibody include the monoclonal antibody produced by the hybridoma having Deposition No. FERM BP-10534 (NAH33 antibody).
  • the ratio is more preferably 100:80 to 100:130, and particularly preferably about 100:110.
  • the antibody preferably achieves a ratio of the reactivity against NAH to the reactivity against about 20% de-N-acetylated NAH of 100:60 to 100:150, and preferably achieves a ratio of the reactivity against NAH to the reactivity against about 10% de-N-acetylated NAH of 100:60 to 100:100.
  • Specific examples of such an antibody include the monoclonal antibody produced by the hybridoma having Deposition No. FERM BP-10535 (NAH43 antibody).
  • an antibody that does not substantially react with about 40% de-N-acetylated NAH preferably achieves a ratio of the reactivity against NAH to the reactivity against about 20% de-N-acetylated NAH of 100:10 to 100:50. Further, the antibody preferably achieves a ratio of the reactivity against NAH to the reactivity against about 10% de-N-acetylated NAH of 100:60 to 100:100. Specific examples of such an antibody include the monoclonal antibody produced by the hybridoma having Deposition No. FERM BP-10536 (NAH46 antibody).
  • de-N-acetylated NAH can be prepared in accordance with the method described in “[Reference Example 1] 2. Preparation of PDNAc-NAH” shown below.
  • the de-N-acetylation ratio (%) of de-N-acetylated NAH means a de-N-acetylation ratio obtained by the method described in “[Reference Example 1] 2. Preparation of PDNAc-NAH” shown below.
  • the antibodies of the present invention can recognize nonsulfated region in NAH and bovine kidney HS.
  • nonsulfated region examples include a region composed of N-acetylglucosamine units or glucosamine units. Such nonsulfated region may comprise either one or both of the N-acetylglucosamine unit and glucosamine unit.
  • the kind of the antibody of the present invention is not particularly limited, and the antibody may be a polyclonal antibody but is preferably a monoclonal antibody.
  • the monoclonal antibody may be a fragment thereof. Examples of the fragment of a monoclonal antibody include F(ab′) 2 antibodies, Fab antibodies, short-chain antibodies (scFv), diabodies, and minibodies.
  • the immunoglobulin class of the monoclonal antibody is not particularly limited, but the class is preferably IgM, and more preferably IgM of ⁇ -chain.
  • the monoclonal antibody can be obtained by, for example, producing a hybridoma by cell fusion of an antibody-producing cells (preferably lymphocytes) of a mammal (preferably a mouse) immunized with, as an antigen, a substance prepared by chemically binding a protein to NAH, and myeloma cells of a mammal (preferably a mouse), selecting a hybridoma that produces a monoclonal antibody having the above-mentioned properties from the resultant hybridoma, and collecting a monoclonal antibody from the culture supernatant of the hybridoma.
  • an antibody-producing cells preferably lymphocytes
  • a mammal preferably a mouse
  • the antigenic substance to be used for producing the antibody is a substance that is obtained by chemically binding a protein to NAH and has the antigenicity that enables production of an antibody that reacts with NAH.
  • the substance can be obtained by, for example, covalently binding NAH prepared by the method described in JP 2004-018840 A to a protein.
  • the first step of obtaining the antibodies of the present invention is to establish novel monoclonal hybridoma capable of producing the antibodies. Specific details of the method of establishing the hybridoma will be described in Examples, and the method briefly includes the following three main steps:
  • NAH is preferably used as an antigen for the immunization step.
  • the NAH is preferably chemically bound to a protein before use.
  • the protein is preferably bovine serum albumin (hereinafter, referred to as “BSA”) or hemocyanin (hereinafter, referred to as “KLH”).
  • GAG or proteoglycan is preferably used as an antigen to be used for immunization, and NAH is more preferably used.
  • they are preferably bound to a protein before use.
  • the mode of chemical bond of a protein and GAG or proteoglycan is not particularly limited, but it is preferably a covalent bond, more preferably a disulfide bond (hereinafter, referred to as “—SS—”).
  • GAG is reductively aminated, and allowed to react with 5 mM N-succinimidyl-3-(2-pyridyldithio)propionate (hereinafter, referred to as “SPDP”), to thereby yield a 2-pyridyldithiopropynylated GAG.
  • SPDP N-succinimidyl-3-(2-pyridyldithio)propionate
  • the resultant 2-pyridyldithiopropynylated GAG is reduced with a reductant such as dithiothreitol to yield thiolated GAG.
  • a protein is allowed to react with SPDP, to thereby yield a 2-pyridyldithiopropynylated protein.
  • GAG antigen a solution of the thiolated GAG and a solution of the 2-pyridyldithiopropynylated protein are mixed to form an SS bond between the GAG and the protein, to thereby yield a GAG-SS-protein (hereinafter, referred to as “GAG antigen”).
  • the animal to be immunized in the present invention is preferably a mammal such as pig, cow, mouse, or rat, and a mouse is particularly preferable because myeloma cells to be used as antibody-producing cells are preferably derived from a mouse.
  • immunization can be performed by subcutaneously-injecting the NAH antigen prepared by the aforementioned method to a mammal.
  • the injection method is not limited thereto, and intraperitoneal injection or intravenous injection may be employed.
  • the immunization is performed in several times, and is preferably performed by administering an antigen together with an adjuvant.
  • the adjuvant may be a substance that is expected to have an adjuvant effect, such as alum, dead tubercle bacilli, nucleic acid, complete Freund's adjuvant, or incomplete Freund's adjuvant, and in particular, TiterMAX Gold (manufactured by Sigma-Aldrich Co.) is preferable.
  • a tumor cell strain to be used for cell fusion is preferably a myeloma cell, and in general, P3-NS-1/1-Ag4-1, P3-X63-Ag8-U1(P3 U1), P3-X63-Ag8-653, SP2/0-Ag14, etc. derived from BALB/c mouse may be used.
  • hybridoma and cloning may be performed by the following method. That is, a hybridoma that produces a desired antibody capable of reacting with NAH is selected from a culture supernatant of proliferating hybridoma by various analysis methods (such as the RIA method, plaque method, agglutination method, ELISA method, flow cytometry method, tissue staining method, and Western blotting method), and then the resultant hybridoma is cloned.
  • the cloning method include fluorescent activated cell sorter (FACS) and a generally used method such as limiting dilution method.
  • the limiting dilution method is preferably performed so that one or less cell is present per well in a 96-well plate.
  • cloning is preferably repeated twice so that an antibody is obtained as a monoclonal antibody.
  • the second step of obtaining the antibodies of the present invention is to produce anti-NAH monoclonal antibodies.
  • Examples of a method of producing an anti-NAH monoclonal antibody include a method of cultivating a resultant monoclonal cell in vitro in large scale and a method of cultivating a resultant monoclonal cell in vivo (in ascites), and the method can be selected depending on the purpose.
  • a monoclonal antibody produced by a single hybridoma can be separated and purified from a cell culture solution or an ascites of a mouse transplanted with the hybridoma. Purification of an antibody from the crude antibody solution can be performed by an appropriate combination of general biochemical techniques such as salting-out, ion exchange, gel filtration, affinity chromatography, and electrophoresis.
  • Examples of a monoclonal antibody obtained as described above include a monoclonal antibody produced by the hybridoma named NAH33 (hereinafter, referred to as NAH33 antibody).
  • NAH33 antibody a monoclonal antibody produced by the hybridoma named NAH33 (hereinafter, referred to as NAH33 antibody).
  • the hybridoma was deposited at the International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Central 6, 1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, 305-8566 Japan) under Deposition No. FERM P-20457 on Mar. 11, 2005. Then, it was transferred to the international deposit under Budapest Treaty under Deposition No. FERM BP-10534.
  • NAH33 antibody has the following reactivity. That is, the antibody reacts with NAH and bovine kidney HS and does not substantially react with mouse EHS-HS, HA, HEP, Ch, CS-A, CS-B, CS-C, CS-D, CS-E, KS, PDNAc-NAH (about 40% de-N-acetylated NAH, about 20% de-N-acetylated NAH, and about 10% de-N-acetylated NAH).
  • the reactivity of the antibody to bovine kidney HS is about 40% as compared to that to NAH. Further, a ratio of the reactivity against NAH to the reactivity against Ac-NAH is about 1:1.
  • NAH43 antibody examples include the monoclonal antibody produced by the hybridoma named NAH43 (hereinafter, referred to as NAH43 antibody).
  • the hybridoma was deposited at the International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Central 6, 1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, 305-8566 Japan) under Deposition No. FERM P-20458 on Mar. 11, 2005. Then, it was transferred to the international deposit under Budapest Treaty under Deposition No. FERM BP-10535.
  • NAH43 antibody has the following reactivity. That is, the antibody reacts with NAH and bovine kidney HS and does not substantially react with mouse EHS-HS, HA, HEP, Ch, CS-A, CS-B, CS-C, CS-D, CS-E, and KS.
  • the reactivity of the antibody to bovine kidney HS is about 75% as compared to that to NAH. There is substantially no difference between the reactivity of the antibody to NAH and the reactivity of the antibody to about 20% de-N-acetylated NAH.
  • a ratio of the reactivity against NAH to the reactivity against about 40% de-N-acetylated NAH and ratio of the reactivity against NAH to the reactivity against about 20% de-N-acetylated NAH are each about 100:110.
  • a ratio of the reactivity against NAH to the reactivity against about 10% de-N-acetylated NAH is about 100:80.
  • the reactivity to Ac-NAH is about 70% as compared to that to NAH.
  • NAH46 antibody examples include the monoclonal antibody produced by the hybridoma named NAH46 (hereinafter, referred to as NAH46 antibody).
  • the hybridoma NAH46 was deposited at the International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Central 6, 1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, 305-8566 Japan) under Deposition No. FERM P-20459 on Mar. 11, 2005. Then, it was transferred to the international deposit under Budapest Treaty under Deposition No. FERM BP-10536.
  • NAH46 antibody has the following reactivity. That is, the antibody reacts with NAH and bovine kidney HS and does not substantially react with mouse EHS-HS, HA, HEP, Ch, CS-A, CS-B, CS-C, CS-D, CS-E, and KS.
  • the reactivity of the antibody to bovine kidney HS is about 40% as compared to that to NAH.
  • the antibody does not substantially react with about 40% de-N-acetylated NAH.
  • the reactivity to about 20% de-N-acetylated NAH is about 40% as compared to that to NAH
  • the reactivity to about 10% de-N-acetylated NAH is about 80% as compared to that to NAH.
  • a ratio of the reactivity against NAH to the reactivity against Ac-NAH is about 1:1.
  • the antibodies of the present invention can be used in detection of HS or NAH present in a sample. Therefore, the present invention provides a method of detecting HS or NAH present in a sample, characterized by using the antibody of the present invention.
  • the sample is not particularly limited as long as it contains or may contain HS or NAH.
  • the phrase “contains HS or NAH” means that a sample may contain either one or both of HA and NAH.
  • the sample is not particularly limited as long as it may contain HS or NAH, and examples of the origin of the sample include body fluids such as urine, blood, plasma, serum, synovial fluid, and marrow, secreted material, cells, tissues, organs, culture supernatants of animal and plant cells or microorganisms, and culture products such as cells themselves or bacterial cells themselves.
  • the term “origin” means that the sample may be a purified product, an extracted product, a specimen, etc. derived from the above-mentioned origins, or may be any of the origins themselves.
  • the detection method of the present invention may be performed using a general immunohistochemical staining method or the like, while in the case of using a body fluid as a sample, the method may be performed by using the ELISA method, RIA method, sandwich method, competition method, plaque method, agglutination method, flow cytometry method, and Western blotting method, or the like.
  • the antibody of the present invention may be immobilized to a solid phase such as a plate as a primary antibody, or the antibody of the present invention may be labeled as a secondary antibody.
  • a solid phase such as a plate as a primary antibody
  • the antibody of the present invention may be labeled as a secondary antibody.
  • the antibody of the present invention include NAH33 antibody, NAH43 antibody, and NAH46 antibody.
  • a secondary antibody in the case of using the antibody of the present invention as a primary antibody, and a primary antibody in the case of using the antibody of the present invention as a secondary antibody are not particularly limited as long as they are capable of binding to HS or NAH to be detected, and examples thereof include antibodies such as mAb JM403 (hereinafter, referred to as “JM403”) and mAb F58-10E4 (hereinafter, referred to as “10E4”).
  • both the primary and secondary antibodies may be the antibodies of the present invention.
  • the antibodies of the present invention may be used in an appropriate combination as the primary and secondary antibodies, and examples of the combination include: a combination of NAH33 antibody as a primary antibody and any one of NAH33 antibody, NAH43 antibody, and NAH46 antibody as a secondary antibody; a combination of NAH43 antibody as a primary antibody and any one of NAH33 antibody, NAH43 antibody, and NAH46 antibody as a secondary antibody; and a combination of NAH46 antibody as a primary antibody and any one of NAH33 antibody, NAH43 antibody, and NAH46 antibody as a secondary antibody.
  • a detection method of the present invention it is possible to specifically detect HS or NAH that is present in a sample and is recognized by the antibody of the present invention by using the specificity of the reaction of the antibody of the present invention.
  • the detection method of the present invention can be applied to various purposes such as judgment of the presence or absence or the degree of canceration or inflammation in cells or tissues, determination of NAH or HA in a culture supernatant, or judgment of the presence or absence or the degree of various diseases, by using the specificity of the reaction of the antibody of the present invention.
  • the present invention provides a kit for detecting HS or NAH present in a sample, which at least comprises the antibody of the present invention.
  • the detection kit of the present invention it is possible to easily implement the detection method of the present invention.
  • the kit “which at least comprises the antibody of the present invention” include a kit comprising the antibody of the present invention itself (including the antibody of the present invention dissolved in a solution or the like) as a component, a kit comprising a solid phase immobilized with the antibody of the present invention as a component, and a kit comprising the antibody of the present invention labeled with an enzyme or the like as a component.
  • the term “sample” has the same meaning as described above on the detection method of the present invention.
  • the detection kit may include, in addition to the antibody of the present invention, a secondary antibody, a reaction buffer, a washing solution, a reaction substrate, an HS standard solution, etc.
  • NAH was prepared by the fermentation method using Escherichia coli K5 (see JP 2004-018840 A). Characteristics of the resultant NAH are shown below.
  • the nucleic acid content determined by EtBr (ethidium bromide) colorimetric determination was found to be 0.09%.
  • the protein content determined by the Lowry method (Lowry J. et al., J. Biol. Chem. 193, 265-275, (1951)) was found to be 2.38%.
  • the number of moles of free amines determined by the 2,4,6-TNBS method was found to be 0.026 ⁇ M/mg.
  • NAH prepared by the above-mentioned method was used to prepare PDNAc-NAH in accordance with the method by Shaklee, P. N. et al. (Biochem. J., 217, 187 (1984)).
  • Glucosamine hydrochloride was used as a standard to calculate a concentration of glucosamine ( ⁇ g/ml) in a sample solution based on fluorescence intensity.
  • NAH is a polysaccharide comprising glucuronic acid and N-acetylglucosamine, so the concentration of N-acetylglucosamine in 50 ⁇ g/ml NAH is approximately 25 ⁇ g/ml. Therefore, a value of the calculated concentration of glucosamine was divided by a value of the approximate concentration of N-acetylglucosamine, and the resultant value was expressed in percentage, to thereby determine the deacetylation degree.
  • the deacetylation degrees for different reaction time of the deacetylation reaction are shown in the following table.
  • Natural GAGs (NAH, HS, HA, HEP, Ch, CS-A(S), CS-A(W), CS-B, CS-C, CS-D, CS-E, and KS) were dissolved in 0.1 M MES buffer (pH 5.5) to prepare 10 mg/ml GAG solutions.
  • NAH is the product prepared in Reference Example 1
  • HEP is a product manufactured by SPL (derived from pig intestines)
  • other GAGs are products manufacture by Seikagaku Corporation as follows: HA (derived from pig skin), Ch (obtained by treating CS-C shown below with acidic methanol), CS-A(S) (derived from sturgeon notochord), CS-A(W) (derived from whale cartilage), CS-B (derived from pig skin), CS-C (derived from shark cartilage), CS-D (derived from shark cartilage), CS-E (derived from squid cartilage), and KS (derived from bovine cornea).
  • HS was bovine kidney HS manufactured by Seikagaku Corporation. Hereinafter, in the examples, it is simply referred to as bovine kidney HS.
  • PDNAc-NAH30, PDNAc-NAH60, PDNAc-NAH120 described in Reference Example 1 were used as starting materials to perform biotin labeling by the same method as described above.
  • 6DSH, 2DSH, and NSH were prepared by the method of Kariya, Y. et al. (Kariya, Y. et al., J. Biochem., 123, 240 (1998)).
  • NH 2 —HEP, NH 2 -2SH, NH 2 -6SH, NH 2 —CDSH, 6DSH, NSH and NAH as starting materials were N-acetylated in accordance with the method of Danishefsky, I., et al. (Danishefsky, I., et al., Methods Carbohydr. Res., 5, 407 (1965)), to thereby yield NAc-HEP, 2SH, 6SH, CDSH, Ac-6DSH, Ac-NSH, and Ac-NAH, respectively.
  • the flowchart of the method of producing the various modified HEPs is shown in FIG. 1 .
  • the disaccharide compositions of mouse EHS-HS and bovine kidney HS were analyzed.
  • the disaccharide compositions represent the ratio of the above-mentioned disaccharides having the above-mentioned specific structures to the amount of unsaturated disaccharides with identifiable structure obtained by the disaccharide composition analysis with enzyme digestion in Test Method 1 shown below (the total mol % of ⁇ DiHS-0S, ⁇ DiHS-NS, ⁇ DiHS-6S, ⁇ DiHS-US, ⁇ DiHS-di(6,N)S, ⁇ DiHS-di(U,N)S, ⁇ DiHS-di(U,6)S, and ⁇ diHS-tri(U,6,N)S) defined as 100%.
  • the numerical values reflect sulfation of a sulfated polysaccharide before digestion.
  • ⁇ diHS-0S ⁇ hexA1 ⁇ 4HexNAc
  • ⁇ diHS-NS ⁇ hexA1 ⁇ 4HexNS
  • ⁇ diHS-6S ⁇ hexA1 ⁇ 4HexNAc(6S)
  • ⁇ diHS-US ⁇ hexA(2S) 1 ⁇ 4HexNAc
  • ⁇ diHS-di(6,N)S ⁇ hexA1 ⁇ 4HexNS(6S)
  • ⁇ diHS-di(U,N)S ⁇ hexA(2S) 1 ⁇ 4HexNS
  • ⁇ diHS-di(U,6)S ⁇ hexA(2S) 1 ⁇ 4HexNAc(6S)
  • ⁇ diHS-tri(U,N,6)S ⁇ hexA(2S) 1 ⁇ 4HexNS(6S)
  • ⁇ hexA, HexNAc, and HexNS represent unsaturated uronic acid, N-acetylhexosamine, and N-sulfated hexosamine, respectively, and the numbers or alphabets shown in the parentheses represent a binding site of a sulfate group.
  • a method of analyzing a substitution site of a sulfate group in a sulfated polysaccharide was performed as follows. That is, each sulfated polysaccharide was enzymatically digested, and the resultant unsaturated disaccharide (the above-mentioned structural formula: Chemical formula 1) was analyzed by high performance liquid chromatography (HPLC) (see “2 ⁇ 8 Structural analysis using glycosaminoglyean degrading enzyme and HPLC in combination” described in Non-patent Document 2). The peak area of each unsaturated disaccharide was calculated, and the peak area with respect to the total area is represented as a percentage.
  • HPLC high performance liquid chromatography
  • Disaccharide composition (%) ⁇ diHS-0S ⁇ diHS-NS ⁇ diHS-6S ⁇ diHS-US ⁇ diHS-di(6,N)S Bovine kidney HS 53 16 11 0.9 6.3 Mouse EHS-HS 33 58 0.3 0.5 1.5 Disaccharide composition (%) ⁇ diHS-di(U,N)S ⁇ diHS-tri(U,6,N)S ⁇ diHS-HS-di(U,6)S NS(total) Bovine kidney HS 7 5.8 N.D 35.1 Mouse EHS-HS 3.1 N.D N.D — “NS(total)” represents a total of ⁇ DiHS-NS, ⁇ DiHS-(6,N)S, ⁇ DiHS-di(U,N)S, and ⁇ DiHS-tri(U,6,N)S. In the above-mentioned table, “N.D” means below the detection limit.
  • N.D means below the detection limit.
  • Streptavidin (manufactured by Vector Laboratories Inc.) was diluted with PBS( ⁇ ) to 20 ⁇ g/ml, and the streptavidin solution was added to a MaxiSorp(registered trademark) 96-well microplate (manufactured by Nalge Nunc International K.K.) at 50 ⁇ l/well, and the plate was stored at 4° C. for 14 to 18 hours to coat the plate uniformly.
  • the plate was washed with PBS( ⁇ ) twice, and then phosphate buffer (PBS( ⁇ ) containing no sodium chloride and no potassium chloride, pH 7.2 to 7.5: hereinafter, referred to as “PB”) containing ApplieDuo (final dilution ratio: 5-fold, manufactured by Seikagaku Corporation) as a blocking substance for blocking a part uncoated with streptavidin and 0.05% ProClin(registered trademark) 300 (manufactured by SUPELCO) as an antiseptic was added thereto, and the plate was allowed to stand at room temperature for two hours. After that, the blocking solution was sufficiently removed, and the plate was dried at 37° C. for two hours, to thereby yield a desired streptavidin-immobilized plate.
  • the plate was packed in an aluminum-laminated bag together with a desiccant, and stored in a refrigerator.
  • the streptavidin-immobilized plate prepared in Reference Example 4 was washed with 300 ⁇ l of PBS( ⁇ ) containing 0.05% polyoxyethylene (20) sorbitan monolaurate (registered trademark, ICI, a product corresponding to Tween 20, available from Wako Pure Chemical Industries, Ltd.) and 0.05% Proclin(registered trademark) 300 as an antiseptic (hereinafter, referred to as “washing solution”) four times, and then the Bi-GAG solution was added at 100 ⁇ L/well, and the plate was allowed to stand at room temperature for 30 minutes, to thereby bind Bi-GAG to the immobilized streptavidin (hereinafter, referred to as “Bi-GAG-immobilized plate).
  • PBS( ⁇ ) containing 0.05% polyoxyethylene (20) sorbitan monolaurate registered trademark, ICI, a product corresponding to Tween 20, available from Wako Pure Chemical Industries, Ltd.
  • Proclin(registered trademark) 300 as an antiseptic
  • NAH prepared in Reference Example 1 was weighed in an amount of 50 mg, and dissolved in 2 ml of an aqueous solution of 2 M ammonium chloride. 25 mg of sodium cyanoborohydrate was added to the solution, and a reductive amination reaction was carried out at 70° C. for two days. Then, 13 mg of sodium cyanoborohydrate was added to the reaction solution, and the reaction was performed under the same condition for two more days. The solution was cooled in an ice bath, and 400 ⁇ l of acetic acid was added to completely stop the reaction.
  • RA-NAH was collected by the solvent precipitation method using double volume of ethanol and washed with ethanol, followed by freeze-drying, to thereby yield 39.5 mg of a freeze-dried product of RA-NAH.
  • PDP-NAH 2-pyridyl Disulfide Propionylated NAH
  • RA-NAH prepared in the item (1) above was weighed in an amount of 10.4 mg, and dissolved in 2 ml of 0.1 M sodium chloride-0.1 M phosphate buffer (pH 7.5). 80 ⁇ l of a solution of 5 mM N-succinimidyl-3-(2-pyridyldithio)propionate (hereinafter, referred to as “SPDP”, Sigma-Aldrich Co.) dissolved in ethanol was added thereto, and the PDP reaction was carried out at room temperature for 30 minutes. The solution was dialyzed against distilled water to remove excessive SPDP, followed by freeze-drying, to thereby yield 9.5 mg of a freeze-dried product of PDP-NAH.
  • SPDP N-succinimidyl-3-(2-pyridyldithio)propionate
  • PDP-NAH prepared in the item (2) above was weighed in an amount of 9.5 mg, and dissolved in 1 ml of 0.1 M sodium chloride-0.1 M sodium acetate buffer (pH 4.5). Dithiothreitol was added to the solution so as to achieve a final concentration of 25 mM to perform reduction reaction at room temperature for 60 minutes.
  • SH-NAH was collected by the solvent precipitation method using double volume of ethanol. The resultant precipitate was washed with ethanol and freeze-dried, to thereby yield 8.3 mg of a freeze-dried product of SH-NAH.
  • the reaction solution was dialyzed against distilled water overnight to remove excessive SPDP, followed by freeze-drying, to thereby yield 104.2 mg of a freeze-dried product of PDP-BSA and 59.4 mg of a freeze-dried product of PDP-KLH.
  • NAH-SS-KLH Disulfide Bond
  • SH-NAH prepared in the item (3) above and PDP-BSA prepared in the item (4) above were used for requirement study of a GAG/protein mix ratio, and as a result, the GAG/protein mix ratio was determined as 2.
  • SH-NAH (1.5 mg) and PDP-KLH (0.75 mg) were dissolved in 1 ml of 0.1 M sodium chloride-0.1 M phosphate buffer (pH 7.5), and the conjugation reaction was performed at room temperature for two hours.
  • the reaction solution was dialyzed against distilled water overnight to remove pyridyl-2-thione generated in the reaction solution, followed by freeze-drying, to thereby yield 1.9 mg of a freeze-dried product of NAH-SS-KLH.
  • the resultant NAH-SS-KLH was used as an NAH antigen.
  • the NAH antigen prepared in “Preparation of NAH antigen ⁇ part 1” in Example 1 was dissolved in a small amount of purified water, and the resultant solution was added to TiterMAX Gold (2 ml, manufactured by Sigma-Aldrich Co.), which was used as an adjuvant, so as to achieve a final concentration of 0.5 mg/ml (antigen solution).
  • the antigen solution was administered subcutaneously to four BALB/C mice (6-week-old female mice, Charles River Laboratories Japan, Inc.) in an amount of 100 ⁇ l/head twice or three times every two weeks. Then, final immunization was performed by administering only the antigen. Three days after the final immunization, the lymph node or spleen was removed, to thereby yield immunized lymphocytes.
  • lymphocytes obtained in the item (1) above and cultured mouse myeloma cells (P3U1, Shima Laboratories Co., Ltd.) were mixed at a rate of 4.4:1 to 5:1, and fused in the presence of 50% polyethyleneglycol 1500 (manufactured by Roche).
  • the fused cells were cultivated in RPMI 1640 (manufactured by Kohjin Bio Co., Ltd) containing 15% fetal calf serum and HAT for about eight days until colonies were amplified. Cloning was performed using a generally used method, i.e., the limiting dilution method.
  • the cells were diluted with the above-mentioned medium so that one or less cell was present in each well of a 96-well microplate, and cultivated. One or two microplates were used for one mouse, to yield a single clone. The cloning was repeated once or twice.
  • NAH-BSA prepared in “2.
  • Preparation of NAH antigen ⁇ part1” in Example 1 was added to a 96-well microplate at 1 ⁇ g/ml in an amount of 50 ⁇ l per well and immobilized. The antigen solution was discarded, and the plate was blocked with Block Ace (Dainippon Sumitomo Pharma Co., Ltd.) diluted 4-fold at room temperature for one hour or at 4° C. overnight.
  • Block Ace Dainippon Sumitomo Pharma Co., Ltd.
  • the six clones selected in the item (3) above were evaluated by secondary screening, i.e., by the ELISA method using biotin-labeled NAH (hereinafter referred to as “Bi-NAH”).
  • the Bi-NAH-immobilized plate produced in Reference Example 5 was washed with the washing solution four times, and a culture supernatant was added to the plate at 100 ⁇ l/well.
  • the plate was allowed to stand at room temperature (15 to 25° C.) for 60 minutes to perform an antigen-antibody reaction. A well containing only the reaction solution was used as a blank. After completion of the reaction, the plate was washed with the washing solution four times, and a secondary antibody solution obtained by diluting horseradish peroxidase-labeled goat anti-mouse immunoglobulin antibody (manufactured by Dako) 2,000-fold with the reaction solution was added at 100 ⁇ l/well.
  • TMB solution tetramethylbenzidine solution
  • a reaction stop solution (manufactured by BioFX Laboratories) was added to the plate at 100 ⁇ l/well to stop the reaction, and absorbance at a wavelength of 450 nm (reference wavelength: 630 nm), which was increased by decomposition of TMB (see Example 4), was determined using a well reader SK-603(registered trademark) (purchased from Seikagaku Corporation). The reactivity of each antibody was evaluated based on the absorbance difference calculated by subtracting the absorbance of the blank from the absorbance of a culture supernatant of each clone for Bi-NAH (hereinafter, referred to as absorbance difference).
  • NAH33, NAH43, and NAH46 clone names: NAH33, NAH43, and NAH46.
  • mice Three pristane-treated BALB/C mice (15-week-old female mice, provided by Charles River Laboratories Japan, Inc.) were used to prepare ascitic fluids containing the clones. To the abdominal cavity of each mouse were transplanted 5 ⁇ 10 6 cells, and the ascitic fluid was collected from the tenth day to the twelfth day several times. The volumes of the ascitic fluids containing the three clones (NAH33, NAH43, and NAH46) were 20 ml, 26 ml, and 40 ml, respectively.
  • the antibody contents in the collected ascitic fluids were evaluated by the cellulose acetate membrane electrophoresis (nitrocellulose membrane (SERECA-V), 60 mM barbital buffer (pH 8.6), 1 mV/cm (width), nigrosine staining).
  • cellulose acetate membrane electrophoresis nitrocellulose membrane (SERECA-V)
  • 60 mM barbital buffer pH 8.6
  • 1 mV/cm width
  • nigrosine staining nigrosine staining
  • the isotypes of the antibodies were examined in accordance with a conventional method, and the isotype of all the three antibodies was found to be IgM ⁇ -chain. Therefore, the antibodies were purified by chromatography using HiTrap IgY Purification HP column (5 ml, Amersham Bioscience). Specifically, the mouse ascitic fluid was dialyzed against a binding buffer (20 mM phosphate buffer, 0.5 M K 2 SO 4 , pH 7.5), followed by filtration using a 0.45 ⁇ m filter. The filtrate was passed through the column sufficiently equilibrated with a binding buffer.
  • the column was washed with the binding buffer until the monitored OD value reached zero, and then an elution buffer (20 mM phosphate buffer, pH 7.5) was passed through the column to elute proteins bound to the column.
  • the eluted protein fractions were collected, and ammonium sulfate powder was added so as to achieve 50% saturation to precipitate proteins.
  • the precipitates were collected by centrifugation, and the resultant product was sufficiently dialyzed against PBS.
  • purified antibodies, NAH33 antibody, NAH43 antibody, and NAH46 antibody were obtained in amounts of 3.4 mg, 0.4 mg, and 3.1 mg, respectively.
  • NAH33 antibody, NAH43 antibody, and NAH46 antibody The reactivity of NAH33 antibody, NAH43 antibody, and NAH46 antibody to various antigens was examined by the ELISA method using Bi-GAG-immobilized plates.
  • test antibody solutions solutions containing the respective test antibodies, which were diluted with the reaction solution A so as to achieve a desired concentration (hereinafter, referred to as “test antibody solutions”), were added at 100 ⁇ l/well.
  • the plate was allowed to stand at room temperature for 60 minutes to perform an antigen-antibody reaction.
  • a well containing only the reaction solution A was used as a blank.
  • TMB solution tetramethylbenzidine solution
  • a reaction stop solution (manufactured by BioFX Laboratories) was added to the plate at 100 ⁇ l/well to stop the reaction, and absorbance at a wavelength of 450 nm (reference wavelength: 630 nm), which was increased by decomposition of TMB, was determined using the well reader SK-603(registered trademark) (purchased from Seikagaku Corporation).
  • the reactivity of each antibody was evaluated based on the absorbance difference calculated by subtracting the absorbance in the case of using a blank (reaction solution A) instead of the test antibody solution from the absorbance in the case where each test antibody solution was allowed to react with Bi-GAG (hereinafter, referred to as absorbance difference).
  • the concentrations of the three antibodies, i.e., NAH33 antibody, NAH43 antibody, and NAH46 antibody, in the test antibody solutions were 0.3 ⁇ g/ml, 0.8 ⁇ g/ml, and 0.04 ⁇ g/ml, respectively, which were given by the previous study on optimal concentrations of the antibodies with regard to the reactivity to Bi-NAH.
  • the concentrations of NAH33 antibody, NAH43 antibody, and NAH46 antibody were 0.2 ⁇ g/ml, 0.5 ⁇ g/ml, and 0.02 ⁇ g/ml, respectively.
  • the reactivity of the three antibodies to various GAGs was determined by the above-mentioned method to examine the specificities.
  • the results of the reactivity of the antibodies to various GAGs are shown in FIG. 2 .
  • the reactivity of the antibodies was evaluated based on absorbance differences ( FIG. 2 ).
  • NAH33 antibody, NAH43 antibody, and NAH46 antibody were found to have reactivity to Bi-NAH and Bi-HS and have no reactivity to Bi-HA, Bi-HEP, Bi-Ch, Bi-CS-A(S), Bi-CS-A(W), Bi-CS-B, Bi-CS-C, Bi-CS-D, Bi-CS-E, and Bi-KS.
  • Mouse EHS-HS prepared by the method described in JP 07-53756 B was used as the NAH analogue.
  • Mouse EHS-HS is a molecule having the same basic skeleton as HS and including D-glucosamine residues in which about 65% of its N-positions are sulfated.
  • the above-mentioned PDNAc-NAH60 was used as a modified NAH.
  • the reactivity of each antibody was evaluated as a relative value, i.e., the reactivity to biotin-labeled EHS-HS (Bi-EHS-HS) or Bi-PDNAc-NAH60 prepared in accordance with the method described in Reference Example 2 with respect to the reactivity to Bi-NAH (absorbance difference) defined as 100%. Determination was performed by the same method as described in “1. Reactivity to various biotin-labeled GAGs (Bi-GAGs)” above. A test on the reactivity to a site-specifically desulfated product obtained from HEP as a starting material, described in “2.
  • FIG. 4 does not show relative values calculated based on the reactivity of NAH but shows absorbance difference.
  • NAH33 antibody reacted with NAH and bovine kidney HS but did not react with EHS-HS and PDNAc-NAH60.
  • This result suggests that acetylated amino groups in a glucosamine residue are important in the reaction of NAH33 antibody to NAH or HS, and glucosamine or N-sulfated glucosamine may act negatively to inhibit the reaction.
  • an antigen determinant in the reaction of NAH33 antibody to NAH or HS includes GlcNAc, and its structure is important for the reaction.
  • NAH43 antibody reacted with NAH, bovine kidney HS, and PDNAc-NAH60, and the reactivity strength was found to be as follows: PDNAc-NAH60 ⁇ NAH>bovine kidney HS.
  • the reactivity to mouse EHS-HS was found to be less than 2% as compared to that to NAH.
  • an antigen determinant in the reaction of NAH43 antibody to NAH or HS includes GlcNAc and GlcNH 2 .
  • the reactivity to mouse EHS-HS was found to be about 5% as compared to that to NAH.
  • an antigen determinant in the reaction of NAH46 antibody to NAH or HS includes GlcNAc, and its structure is important for the reaction.
  • Bi-NAH, Bi-HS, Bi-PDNAc-NAH60 were decomposed with heparitinase (manufacture by Seikagaku Corporation) to examine the decrease in or disappearance of the reactivity of the antibodies.
  • Heparitinase solution was prepared to 50 mU/mL with 50 mM Tris (manufactured by Sigma-Aldrich Co.)-HCl buffer (pH 7.3 to 7.7) containing ApplieDuo® (final dilution ratio: 20-fold, manufactured by Seikagaku Corporation), 0.15 M sodium chloride (manufactured by Nacalai Tesque, Inc.), 5 mM calcium chloride (manufactured by Wako Pure Chemical Industries, Ltd.), and 0.05% ProClin 300 as an antiseptic (hereinafter, referred to as “diluted heparitinase solution”).
  • the Bi-GAG-immobilized plate and Bi-PDNAc-NAH60-immobilized plate produced as the aforementioned examples were washed with the washing solution four times, and the diluted heparitinase solution was added at 100 ⁇ L/well.
  • the plates were allowed to stand at 37° C. for two hours to digest immobilized Bi-GAG and immobilized Bi-PDNAc-NAH60. After digestion, the plates were washed in the same way as described above, and the three kinds of antibodies were added in accordance with the method described in the above-described example. Then, subsequent procedures were performed to determine the reactivity. The reactivity was evaluated based on absorbance difference. The results are shown in FIGS. 5(A) to (C).
  • each antibody was analyzed by the same method as Example 4 except that the concentrations of Bi-GAGs were changed within the range of 0.0 to 1.0 ⁇ g/mL.
  • PDNAc-NAH30, PDNAc-NAH60, PDNAc-NAH120, described in Reference Example 1 were used as the Bi-PDNAc-NAHs.
  • the results are shown in FIGS. 6( a ) to ( f ).
  • the results for the GAGs, described in “2. Biotin labeling of chemically-modified GAG”, are shown in FIGS. 7( a ) to ( c ).
  • the resultant sections were air-dried for two hours at room temperature and fixed with cold acetone (4° C.), followed by air-drying at room temperature for one hour. After that, the sections were washed with PBS( ⁇ ) and immersed in distilled water containing 0.1% sodium azide (manufactured by Wako Pure Chemical Industries, Ltd.) and 0.3% hydrogen peroxide solution (manufactured by Wako Pure Chemical Industries, Ltd.) at room temperature for 10 minutes to remove endogenous peroxidase activity, and then the sections were washed with PBS( ⁇ ) and blocked with PBS( ⁇ ) containing 0.1% casein (manufactured by Wako Pure Chemical Industries, Ltd.) (hereinafter, referred to as “PBS”) at room temperature for 60 minutes.
  • PBS casein
  • the sections were washed with PBS( ⁇ ), and NAH33 antibody, NAH43 antibody, and NAH46 antibody were diluted with PBS to 0.5 ⁇ g/mL, 2 ⁇ g/mL, and 0.5 ⁇ g/mL, respectively, and were allowed to react at 4° C. overnight.
  • the sections were washed with PBS( ⁇ ), and biotin-labeled anti-mouse IgG+IgM containing 10% rat serum (manufactured by The Jackson Laboratories) was diluted 500-fold with PBS, followed by reaction at room temperature for 30 minutes.
  • the sections were immersed in PBS( ⁇ ) to stop the reaction, and were washed with water for five minutes. After that, nuclear staining (blue) was performed with hematoxylin (manufactured by DAKO) for comparative staining. The sections were washed with water for five minutes, and dehydration and penetration procedures were performed in accordance with a conventional method, followed by embedding.
  • FIG. 8 Staining images are shown in FIG. 8 .
  • brown strong positive reactions (arrowed lines) were observed in the renal glomerular basement membrane, vascular endothelium, and Bowman's capsule, while in the case of NAH43, positive reactions (arrowhead) were observed in the renal glomerular basement membrane and vascular endothelium.
  • Sections were prepared by the above-mentioned method, and were fixed with cold acetone (4° C.), followed by air-drying.
  • the sections were washed with PBS( ⁇ ), and heparitinase I (manufactured by Seikagaku Corporation) was diluted to 250 mU/ml with 20 mM acetic acid (manufactured by Wako Pure Chemical Industries, Ltd.) buffer (pH 7.0) containing 1 ⁇ M calcium acetate (manufactured by Wako Pure Chemical Industries, Ltd.) (hereinafter, referred to as “buffer”) and allowed to react at 37° C. for two hours.
  • buffer pH 7.0
  • As a control only the buffer was allowed to react in the same way as described above.
  • the sections were washed with PBS( ⁇ ), and the antibodies were allowed to react in the same way as described above, followed by the brown color development reaction using the DAB color development kit (manufactured by Zymed Laboratories Inc.).
  • the sections were immersed in PBS( ⁇ ) to stop the reaction, and washed with water for five minutes. After that, nuclear staining (blue) was performed with hematoxylin (manufactured by DAKO) for comparative staining. The sections were washed with water for five minutes, and dehydration and penetration procedures were performed in accordance with a conventional method, followed by embedding.
  • NAH46 antibody was used to detect bovine kidney HS using an antigen-immobilized plate.
  • NAH46 antibody was diluted to 20 ⁇ g/ml with PBS( ⁇ ), and the antibody solution was added to a MaxiSorp (registered trademark) 96-well microplate at 50 ⁇ l/well, and the plate was stored at 4° C. for 14 to 18 hours to coat the plate uniformly.
  • the plate was washed with PBS( ⁇ ) twice, and phosphate buffer (PBS( ⁇ ) containing no sodium chloride and no potassium chloride, pH 7.2 to 7.5) containing ApplieDuo (final dilution ratio: 5-fold) as a blocking substance and 0.05% ProClin (registered trademark) 300 as an antiseptic was added thereto, and the plate was allowed to stand at room temperature for two hours.
  • PBS( ⁇ ) phosphate buffer
  • ApplieDuo final dilution ratio: 5-fold
  • ProClin registered trademark
  • NAH46 antibody-immobilized plate was packed in an aluminum-laminated bag together with a desiccant, and stored in a refrigerator.
  • JM403 and 10E4 both manufactured by Seikagaku Corporation were used instead of NAH46 antibody, to thereby prepare a JM403-immobilized plate and a 10E4-immobilized plate.
  • the NAH46 antibody solution (1 mg/ml) was labeled with HRP using a commercially available HRP labeling kit (manufactured by Dojindo Laboratories) by a designated method.
  • the resultant HRP-labeled antibody was diluted 800-fold with a solution containing ApplieDuo (final dilution ratio: 20-fold), pH 7.3 to 7.7 Tris-HCl (50 mM), NaCl (8 g/L), Tween 20 (0.05%), and ProClin 300 (0.05%) as an antiseptic (hereinafter, referred to as “reaction solution for measurement”), to thereby prepare an HRP-labeled antibody solution.
  • reaction solution for measurement was added to the antibody-immobilized plates described in the item (1) above at 100 ⁇ l/well. Subsequently, solutions of bovine kidney HS, prepared to about 10.0, 5.0, 2.5, 1.25, 0.63, 0.31, 0.16 ⁇ g/ml with the reaction solution for measurement, were added at 20 ⁇ l/well, and the plates were allowed to stand at 4° C. for 18 hours, to thereby form complexes of “immobilized antibody-bovine kidney HS” (first reaction). A well containing only the reaction solution for measurement (bovine kidney HS; 0 ⁇ g/ml) was used as a blank.
  • the plates were washed four times with a washing solution (a solution containing pH 7.3 to 7.7 Tris-HCl (50 mM), NaCl (8 g/L), Tween 20 (0.05%), and ProClin 300 (0.05%)) at 300 ⁇ l/well.
  • a washing solution a solution containing pH 7.3 to 7.7 Tris-HCl (50 mM), NaCl (8 g/L), Tween 20 (0.05%), and ProClin 300 (0.05%)
  • the HRP-labeled antibody solutions prepared in the item (2) above were added at 100 ⁇ l/well in the combinations as shown in Table 4 below, and the plates were allowed to stand at 4° C. for three hours, to thereby form complexes of “immobilized antibody-bovine kidney HS-HRP-labeled antibody” (second reaction).
  • FCM flow cytometry method
  • colo201 cells (derived from human colon cancer, purchased from Dainippon Sumitomo Pharma Co., Ltd.) were suspended in a medium, and cultivated in a CO 2 incubator (manufactured by Ikemoto Scientific Technology Co., Ltd., in the presence of 5% CO 2 , 37° C.) for three days.
  • the medium was prepared by adding to RPMI 1640 with L-glutamine (manufactured by Sigma-Aldrich Co.) the heat-inactivated fetal calf serum (manufactured by ICN Biomedicals, Inc.) at a concentration of 10% and penicillin-streptomycin (Dainippon Sumitomo Pharma Co., Ltd.) so that the concentration of penicillin G sodium is 60 IU/mL and the concentration of streptomycin sulfate is 60 ⁇ g/mL.
  • the resultant cultivated cells were suspended in PBS( ⁇ ) to 10 6 cells/mL.
  • the HS-positive cells and HS-negative cells obtained in the section (1) above were separately subjected to washing by centrifugation (1,500 rpm, 5 minutes, 4° C.) with PBS( ⁇ ) twice and suspended in a solution of 10 ⁇ g/mL of NAH46 antibody, followed by reaction under shading at 4° C. for 30 minutes.
  • the resultant cells were subjected to washing by centrifugation twice and allowed to react with FITC-labeled anti-mouse IgG+IgM (manufactured by The Jackson Laboratories) diluted 50-fold with PBS( ⁇ ) under shading at 4° C. for 30 minutes.
  • the resultant cells were further subjected to washing by centrifugation twice and suspended in 1 mL of PBS( ⁇ ), and the suspension was filtered using a tube equipped with a filter (manufactured by Becton, Dickinson and Company).
  • the resultant cell suspensions were analyzed using a flow cytometer EPICS XL-MCL (manufactured by Beckman Coulter, Inc.).
  • mouse IgM manufactured by Sigma-Aldrich Co.
  • NAH46 antibody the NAH46 antibody
  • mouse IgM is referred to as a negative control antibody.
  • the negative control antibody did not react with the HS-positive cells (FIG. 12 (A)), while NAH46 antibody reacted with the HS-positive cells ( FIG. 12(B) ) and detected HS on the surfaces of colo201 cells.
  • the negative-control antibody and NAH46 antibody did not react with the HS-negative cells ( FIG. 12(C) ).
  • the antibody of the present invention can specifically detect HS on the surfaces of cells.
  • the antibodies of the present invention reacts with NAH and bovine kidney HS and therefore can be suitably used for the detection of HS or NAH in a sample. Meanwhile, the antibodies of the present invention do not substantially react with mouse ESH-HS and therefore can specifically detect HS of low sulfation degree. In addition, the antibodies of the present invention can specifically recognize nonsulfated region of HS, which is composed of N-acetylglucosamine units or glucosamine units, and therefore the antibodies of the present invention can be applied to specific detection of the above-mentioned structures.

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JP5467185B2 (ja) * 2008-01-31 2014-04-09 愛知県 アポトーシスの検出方法
CA2773755C (en) * 2008-09-09 2017-04-25 The Board Of Regents Of The University Of Oklahoma Heparosan polymers and methods of making and using same for the enhancement of therapeutics
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CN103149198B (zh) * 2013-02-08 2015-04-22 浙江工业大学 一种酶法快速定量检测heparosan的方法
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US20100036001A1 (en) * 2008-03-19 2010-02-11 Deangelis Paul L Heparosan polymers and methods of making and using same for the enhancement of therapeutics
US9603945B2 (en) 2008-03-19 2017-03-28 The Board Of Regents Of The University Of Oklahoma Heparosan polymers and methods of making and using same for the enhancement of therapeutics
US9687559B2 (en) * 2008-03-19 2017-06-27 The Board Of Regents Of The University Of Oklahoma Heparosan polymers and methods of making and using same for the enhancement of therapeutics
US9925209B2 (en) 2008-03-19 2018-03-27 The Board Of Regents Of The University Of Oklahoma Heparosan-polypeptide and heparosan-polynucleotide drug conjugates and methods of making and using same

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